Abstract

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Abstract

Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients.

Abstract

The primary cilium is an antenna-like cellular protrusion mediating sensory and neuroendocrine signaling. Its localization within tissue architecture and a growing list of cilia-localized receptors, in particular G-protein-coupled receptors, determine a host of crucial physiologies, which are disrupted in human ciliopathies. Here, we discuss recent advances in the identification and characterization of ciliary signaling components and pathways. Recent studies have highlighted the unique signaling environment of the primary cilium and we are just beginning to understand how this design allows for highly amplified and regulated signaling.

Abstract

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment.

Abstract

Short-rib polydactyly syndromes (SRPS) arise from mutations in genes involved in retrograde intraflagellar transport (IFT) and basal body homeostasis, which are critical for cilia assembly and function. Recently, mutations in WDR34 or WDR60 (candidate dynein intermediate chains) were identified in SRPS. We have identified and characterized Tctex1d2, which associates with Wdr34, Wdr60 and other dynein complex 1 and 2 subunits. Tctex1d2 and Wdr60 localize to the base of the cilium and their depletion causes defects in ciliogenesis. We propose that Tctex1d2 is a novel dynein light chain important for trafficking to the cilium and potentially retrograde IFT and is a new molecular link to understanding SRPS pathology.

Abstract

We have developed a novel 3D cell culture model that uses mouse inner-medullary collecting duct (mIMCD3) cells to generate epithelial spheroids. This model is amenable to efficient siRNA knockdown and subsequent rescue with human patient-derived alleles. Spheroids develop apicobasal polarity and complete lumens, and they are consequently an ideal model for polarity defects seen in renal ciliopathies such as nephronophthisis. Briefly, mIMCD3 cells are transfected and subsequently passaged to a Matrigel mixture, which is seeded in chamber slides and covered in growth medium. Once the spheroids are formed, Matrigel is dissolved and immunocytochemistry is performed in the chamber slides. The technique is amenable to semiautomatic imaging analysis, and it can test multiple genes simultaneously, gene-dosing effects and a variety of therapeutic interventions. The spheroid technique is a unique and simple 6-d in vitro method of interrogating ex vivo tissue organization.

Abstract

Activation of Checkpoint kinase 1 (Chk1) following DNA damage mediates cell cycle arrest to prevent cells with damaged DNA from entering mitosis. Here we provide a high-resolution analysis of cells as they undergo S- and G₂-checkpoint bypass in response to Chk1 inhibition with the selective Chk1 inhibitor GNE-783. Within 4-8 h of Chk1 inhibition following gemcitabine induced DNA damage, cells with both sub-4N and 4N DNA content prematurely enter mitosis. Coincident with premature transition into mitosis, levels of DNA damage dramatically increase and chromosomes condense and attempt to align along the metaphase plate. Despite an attempt to congress at the metaphase plate, chromosomes rapidly fragment and lose connection to the spindle microtubules. Gemcitabine mediated DNA damage promotes the formation of Rad51 foci; however, while Chk1 inhibition does not disrupt Rad51 foci that are formed in response to gemcitabine, these foci are lost as cells progress into mitosis. Premature entry into mitosis requires the Aurora, Cdk1/2 and Plk1 kinases and even though caspase-2 and -3 are activated upon mitotic exit, they are not required for cell death. Interestingly, p53, but not p21, deficiency enables checkpoint bypass and chemo-potentiation. Finally, we uncover a differential role for the Wee-1 checkpoint kinase in response to DNA damage, as Wee-1, but not Chk1, plays a more prominent role in the maintenance of S- and G₂-checkpoints in p53 proficient cells.

Abstract

Nicotinamide adenine dinucleotide (NAD) is a metabolite essential for cell survival and generated de novo from tryptophan or recycled from nicotinamide (NAM) through the nicotinamide phosphoribosyltransferase (NAMPT)-dependent salvage pathway. Alternatively, nicotinic acid (NA) is metabolized to NAD through the nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1)-dependent salvage pathway. Tumor cells are more reliant on the NAMPT salvage pathway making this enzyme an attractive therapeutic target. Moreover, the therapeutic index of NAMPT inhibitors may be increased by in NAPRT-deficient tumors by NA supplementation as normal tissues may regenerate NAD through NAPRT1. To confirm the latter, we tested novel NAMPT inhibitors, GNE-617 and GNE-618, in cell culture- and patient-derived tumor models. While NA did not protect NAPRT1-deficient tumor cell lines from NAMPT inhibition in vitro, it rescued efficacy of GNE-617 and GNE-618 in cell culture- and patient-derived tumor xenografts in vivo. NA co-treatment increased NAD and NAM levels in NAPRT1-deficient tumors to levels that sustained growth in vivo. Furthermore, NAM co-administration with GNE-617 led to increased tumor NAD levels and rescued in vivo efficacy as well. Importantly, tumor xenografts remained NAPRT1-deficient in the presence of NA, indicating that the NAPRT1-dependent pathway is not reactivated. Protection of NAPRT1-deficient tumors in vivo may be due to increased circulating levels of metabolites generated by mouse liver, in response to NA or through competitive reactivation of NAMPT by NAM. Our results have important implications for the development of NAMPT inhibitors when considering NA co-treatment as a rescue strategy.

Abstract

Here we report that GNE-783, a novel checkpoint kinase-1 (CHK1) inhibitor, enhances the activity of gemcitabine by disabling the S- and G2 cell-cycle checkpoints following DNA damage. Using a focused library of 51 DNA-damaging agents, we undertook a systematic screen using three different cell lines to determine which chemotherapeutics have their activity enhanced when combined with GNE-783. We found that GNE-783 was most effective at enhancing activity of antimetabolite-based DNA-damaging agents; however, there was a surprisingly wide range of activity within each class of agents. We, next, selected six different therapeutic agents and screened these in combination with GNE-783 across a panel of cell lines. This revealed a preference for enhanced chemopotentiation of select agents within tumor types, as, for instance, GNE-783 preferentially enhanced the activity of temozolomide only in melanoma cell lines. Additionally, although p53 mutant status was important for the overall response to combinations with some agents; our data indicate that this alone was insufficient to predict synergy. We finally compared the ability of a structurally related CHK1 inhibitor, GNE-900, to enhance the in vivo activity of gemcitabine, CPT-11, and temozolomide in xenograft models. GNE-900 significantly enhanced activity of only gemcitabine in vivo, suggesting that strong chemopotentiation in vitro can translate into chemopotentiation in vivo. In conclusion, our results show that selection of an appropriate agent to combine with a CHK1 inhibitor needs to be carefully evaluated in the context of the genetic background and tumor type in which it will be used.

Abstract

VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.

Abstract

CONTRIBUTING REVIEWERS: We would like to thank all our reviewers for their contribution to the success of Cilia. Their voluntary participation has led to the launch of a new journal consistently publishing high quality articles and an intelligent peer review process for all our authors.

Abstract

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

Abstract

During cell division, cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule copurifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential antimitotic target.

Abstract

The membrane of the primary cilium is a highly specialized compartment that organizes proteins to achieve spatially ordered signaling. Disrupting ciliary organization leads to diseases called ciliopathies, with phenotypes ranging from retinal degeneration and cystic kidneys to neural tube defects. How proteins are selectively transported to and organized in the primary cilium remains unclear. Using a proteomic approach, we identified the ARL3 effector UNC119 as a binding partner of the myristoylated ciliopathy protein nephrocystin-3 (NPHP3). We mapped UNC119 binding to the N-terminal 200 residues of NPHP3 and found the interaction requires myristoylation. Creating directed mutants predicted from a structural model of the UNC119-myristate complex, we identified highly conserved phenylalanines within a hydrophobic β sandwich to be essential for myristate binding. Furthermore, we found that binding of ARL3-GTP serves to release myristoylated cargo from UNC119. Finally, we showed that ARL3, UNC119b (but not UNC119a), and the ARL3 GAP Retinitis Pigmentosa 2 (RP2) are required for NPHP3 ciliary targeting and that targeting requires UNC119b myristoyl-binding activity. Our results uncover a selective, membrane targeting GTPase cycle that delivers myristoylated proteins to the ciliary membrane and suggest that other myristoylated proteins may be similarly targeted to specialized membrane domains.

Abstract

Oncogenic mutations in the mitogen activated protein kinase (MAPK) pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2). We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells.

Abstract

Cell cycle progression requires the E3 ubiquitin ligase anaphase-promoting complex (APC/C), which uses the substrate adaptors CDC20 and CDH1 to target proteins for proteasomal degradation. The APC(CDH1) substrate cyclin A is critical for the G1/S transition and, paradoxically, accumulates even when APC(CDH1) is active. We show that the deubiquitinase USP37 binds CDH1 and removes degradative polyubiquitin from cyclin A. USP37 was induced by E2F transcription factors in G1, peaked at G1/S, and was degraded in late mitosis. Phosphorylation of USP37 by CDK2 stimulated its full activity. USP37 overexpression caused premature cyclin A accumulation in G1 and accelerated S phase entry, whereas USP37 knockdown delayed these events. USP37 was inactive in mitosis because it was no longer phosphorylated by CDK2. Indeed, it switched from an antagonist to a substrate of APC(CDH1) and was modified with degradative K11-linked polyubiquitin.

Abstract

Two papers in this issue show that dynein-binding proteins may regulate the G1-S transition through an effect on cilia. Nde1, a known partner of dynein light chain LC8, controls ciliary length in vitro and in zebrafish, and influences the G1-S progression. The phosphorylation of Tctex1, a dynein light chain, modulates cilia length and accelerates G1-S, thereby regulating proliferation-differentiation decisions in the developing mouse neocortex.

Abstract

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.

Abstract

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.

Abstract

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.

Abstract

The tubby mouse shows a tripartite syndrome characterized by maturity-onset obesity, blindness and deafness. The causative gene Tub is the founding member of a family of related proteins present throughout the animal and plant kingdoms, each characterized by a signature carboxy-terminal tubby domain. This domain consists of a β barrel enclosing a central α helix and binds selectively to specific membrane phosphoinositides. The vertebrate family of tubby-like proteins (TULPs) includes the founding member TUB and the related TULPs, TULP1 to TULP4. Tulp1 is expressed in the retina and mutations in TULP1 cause retinitis pigmentosa in humans; Tulp3 is expressed ubiquitously in the mouse embryo and is important in sonic hedgehog (Shh)-mediated dorso-ventral patterning of the spinal cord. The amino terminus of these proteins is diverse and directs distinct functions. In the best-characterized example, the TULP3 amino terminus binds to the IFT-A complex, a complex important in intraflagellar transport in the primary cilia, through a short conserved domain. Thus, the tubby family proteins seem to serve as bipartite bridges through their phosphoinositide-binding tubby and unique amino-terminal functional domains, coordinating multiple signaling pathways, including ciliary G-protein-coupled receptor trafficking and Shh signaling. Molecular studies on this functionally diverse protein family are beginning to provide us with remarkable insights into the tubby-mouse syndrome and other related diseases.

Abstract

The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C(Cdc20) is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C(Cdc20) specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.

Abstract

Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects. The tubby mouse, which presents with adult-onset obesity, arises from mutation in the Tub gene. The tubby-like proteins comprise a related family of poorly understood proteins with roles in neural development and function. We find that specific Tubby family proteins, notably Tubby-like protein 3 (TULP3), bind to the IFT-A complex. IFT-A is linked to retrograde ciliary transport, but, surprisingly, we find that the IFT-A complex has a second role directing ciliary entry of TULP3. TULP3 and IFT-A, in turn, promote trafficking of a subset of G protein-coupled receptors (GPCRs), but not Smoothened, to cilia. Both IFT-A and membrane phosphoinositide-binding properties of TULP3 are required for ciliary GPCR localization. TULP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3-IFT-A interaction suggests how these proteins cooperate during neural tube patterning.

Abstract

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders that feature dysplasia or degeneration occurring preferentially in the kidney, retina and cerebellum. Here we combined homozygosity mapping with candidate gene analysis by performing 'ciliopathy candidate exome capture' followed by massively parallel sequencing. We identified 12 different truncating mutations of SDCCAG8 (serologically defined colon cancer antigen 8, also known as CCCAP) in 10 families affected by NPHP-RC. We show that SDCCAG8 is localized at both centrioles and interacts directly with OFD1 (oral-facial-digital syndrome 1), which is associated with NPHP-RC. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in three-dimensional renal cell cultures. This work identifies loss of SDCCAG8 function as a cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.

Abstract

Taxanes are very effective at causing mitotic arrest; however, there is variability among cancer cells in the apoptotic response to mitotic arrest. The variability in clinical efficacy of taxane-based therapy is likely a reflection of this variability in apoptotic response, thus elucidation of the molecular mechanism of the apoptotic response to mitotic stress could lead to improved clinical strategies. To identify genes whose expression influences the rate and extent of apoptosis after mitotic arrest, we screened a kinase-enriched small interfering RNA library for effects on caspase activation in response to maximally effective doses of paclitaxel, a PLK1 inhibitor, or cisplatin. Small interfering RNA oligonucleotides directed against an atypical protein kinase, TP53RK, caused the greatest increase in caspase-3/7 activation in response to antimitotic agents. Time-lapse microscopy revealed that cells entered mitosis with normal kinetics, but died after entry into mitosis in the presence of paclitaxel more rapidly when TP53RK was depleted. Because expression levels of TP53RK vary in cancers, TP53RK levels could provide a molecular marker to predict response to antimitotic agents. TP53RK inhibition may also sensitize cancers to taxanes.

Abstract

In early mitosis, the END (Emi1/NuMA/Dynein-dynactin) network anchors the anaphase-promoting complex/cyclosome (APC/C) to the mitotic spindle and poles. Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity. Emi1 binds directly to hypophosphorylated APC/C, linking the APC/C to the spindle via NuMA. However, whether the phosphorylation state of the APC/C is important for its association with the spindle and what kinases and phosphatases are necessary for regulating this event remain unknown. Here, we describe the regulation of APC/C-mitotic spindle pole association by phosphorylation. We find that only hypophosphorylated APC/C associates with microtubule asters, suggesting that phosphatases are important. Indeed, a specific form of PPP2 (CA/R1A/R2B) binds APC/C, and PPP2 activity is necessary for Cdc27 dephosphorylation. Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle. Consistently, inhibition of cyclin B/Cdk1 activity increased APC/C binding to microtubules. Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation.

Abstract

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

Abstract

Deubiquitinating enzymes (DUBs) act on ubiquitinated substrates to regulate their modification and stability. In this issue, Sowa et al. (2009) present a comprehensive proteomic analysis of DUB interacting proteins in humans and a new quantitative scoring system for hits (CompPASS), providing a resource that links DUBs to biological pathways.

Abstract

We present an optimized system for rapid generation of localization and affinity purification-tagged mammalian stable cell lines that facilitates complex purification and interacting protein identification. The improved components of this method, including the flexibility of inducible expression, circumvent issues associated with toxicity, clonal selection, sample yields and time to data acquisition. We have applied this method to the study of cell-cycle regulators and novel microtubule-associated proteins.

Abstract

The Anaphase Promoting Complex (APC) ubiquitin ligase is critical for multiple processes including cell cycle, development, meiosis, and senescence. The importance of regulation of the APC by substrate competitive (pseudosubstrate) inhibitors, such as Emi1 and BubR1, has recently been demonstrated. Substrate competitive inhibitors typically bind to enzymes via the same site as substrates, but by having any combination of increased enzyme affinity and low turnover numbers, are able to "clog" the ability of the enzyme to bind and turnover substrates. For the APC, these pseudosubstrates can both position and block the APC and have been well validated as critical regulators for the APC enzymes.We have found that the substrate competitive mechanism of inhibition is sensitive to the E2 activity driving APC catalyzed ubiquitination events. This chapter provides detailed protocols for multiple in vitro ubiquitination assays of increasing complexity and the use of pseudosubstrate inhibitors in these assays. These assays are instrumental in examining the use of E2 enzymes by the APC and the intimate relationship this has with pseudosubstrate inhibition.

Abstract

Primary cilium dysfunction affects the development and homeostasis of many organs in Bardet-Biedl syndrome (BBS). We recently showed that seven highly conserved BBS proteins form a stable complex, the BBSome, that functions in membrane trafficking to and inside the primary cilium. We have now discovered a BBSome subunit that we named BBIP10. Similar to other BBSome subunits, BBIP10 localizes to the primary cilium, BBIP10 is present exclusively in ciliated organisms, and depletion of BBIP10 yields characteristic BBS phenotypes in zebrafish. Unexpectedly, BBIP10 is required for cytoplasmic microtubule polymerization and acetylation, two functions not shared with any other BBSome subunits. Strikingly, inhibition of the tubulin deacetylase HDAC6 restores microtubule acetylation in BBIP10-depleted cells, and BBIP10 physically interacts with HDAC6. BBSome-bound BBIP10 may therefore function to couple acetylation of axonemal microtubules and ciliary membrane growth.

Abstract

Named originally for their effects on peripheral end organs, the melanocortin system controls a diverse set of physiological processes through a series of five G-protein-coupled receptors and several sets of small peptide ligands. The central melanocortin system plays an essential role in homeostatic regulation of body weight, in which two alternative ligands, alpha-melanocyte-stimulating hormone and agouti-related protein, stimulate and inhibit receptor signaling in several key brain regions that ultimately affect food intake and energy expenditure. Much of what we know about the relationship between central melanocortin signaling and body weight regulation stems from genetic studies. Comparative genomic studies indicate that melanocortin receptors used for controlling pigmentation and body weight regulation existed more than 500 million years ago in primitive vertebrates, but that fine-grained control of melanocortin receptors through neuropeptides and endogenous antagonists developed more recently. Recent studies based on dog coat-color genetics revealed a new class of melanocortin ligands, the beta-defensins, which reveal the potential for cross talk between the melanocortin and the immune systems.

Abstract

In vitro, the anaphase-promoting complex (APC) E3 ligase functions with E2 ubiquitin-conjugating enzymes of the E2-C and Ubc4/5 families to ubiquitinate substrates. However, only the use of the E2-C family, notably UbcH10, is genetically well validated. Here, we biochemically demonstrate preferential use of UbcH10 by the APC, specified by the E2 core domain. Importantly, an additional E2-E3 interaction mediated by the N-terminal extension of UbcH10 regulates APC activity. Mutating the highly conserved N terminus increases substrate ubiquitination and the number of substrate lysines targeted, allows ubiquitination of APC substrates lacking their destruction boxes, increases resistance to the APC inhibitors Emi1 and BubR1 in vitro, and bypasses the spindle checkpoint in vivo. Fusion of the UbcH10 N terminus to UbcH5 restricts ubiquitination activity but does not direct specific interactions with the APC. Thus, UbcH10 combines a specific E2-E3 interface and regulation via its N-terminal extension to limit APC activity for substrate selection and checkpoint control.

Abstract

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B's subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14B(Delta/Delta) cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.

Abstract

Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.

Abstract

The gut epithelium self-renews every several days, providing an important innate defense system that limits bacterial colonization. Nevertheless, many bacterial pathogens, including Shigella, efficiently colonize the intestinal epithelium. Here, we show that the Shigella effector IpaB, when delivered into epithelial cells, causes cell-cycle arrest by targeting Mad2L2, an anaphase-promoting complex/cyclosome (APC) inhibitor. Cyclin B1 ubiquitination assays revealed that APC undergoes unscheduled activation due to IpaB interaction with the APC inhibitor Mad2L2. Synchronized HeLa cells infected with Shigella failed to accumulate Cyclin B1, Cdc20, and Plk1, causing cell-cycle arrest at the G2/M phase in an IpaB/Mad2L2-dependent manner. IpaB/Mad2L2-dependent cell-cycle arrest by Shigella infection was also demonstrated in rabbit intestinal crypt progenitors, and the IpaB-mediated arrest contributed to efficient colonization of the host cells. These results strongly indicate that Shigella employ special tactics to influence epithelial renewal in order to promote bacterial colonization of intestinal epithelium.

Abstract

Overexpression of cyclin E, an activator of cyclin-dependent kinase 2, has been linked to human cancer. In cell culture models, the forced expression of cyclin E leads to aneuploidy and polyploidy, which is consistent with a direct role of cyclin E overexpression in tumorigenesis. In this study, we show that the overexpression of cyclin E has a direct effect on progression through the latter stages of mitotic prometaphase before the complete alignment of chromosomes at the metaphase plate. In some cases, such cells fail to divide chromosomes, resulting in polyploidy. In others, cells proceed to anaphase without the complete alignment of chromosomes. These phenotypes can be explained by an ability of overexpressed cyclin E to inhibit residual anaphase-promoting complex (APC(Cdh1)) activity that persists as cells progress up to and through the early stages of mitosis, resulting in the abnormal accumulation of APC(Cdh1) substrates as cells enter mitosis. We further show that the accumulation of securin and cyclin B1 can account for the cyclin E-mediated mitotic phenotype.

Abstract

Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. Here we describe a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction.

Abstract

Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.

Abstract

The fidelity of cell division is dependent on the accumulation and ordered destruction of critical protein regulators. By triggering the appropriately timed, ubiquitin-dependent proteolysis of the mitotic regulatory proteins securin, cyclin B, aurora A kinase, and polo-like kinase 1, the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays an essential role in maintaining genomic stability. Misexpression of these APC/C substrates, individually, has been implicated in genomic instability and cancer. However, no comprehensive survey of the extent of their misregulation in tumors has been performed. Here, we analyzed more than 1600 benign and malignant tumors by immunohistochemical staining of tissue microarrays and found frequent overexpression of securin, polo-like kinase 1, aurora A, and Skp2 in malignant tumors. Positive and negative APC/C regulators, Cdh1 and Emi1, respectively, were also more strongly expressed in malignant versus benign tumors. Clustering and statistical analysis supports the finding that malignant tumors generally show broad misregulation of mitotic APC/C substrates not seen in benign tumors, suggesting that a "mitotic profile" in tumors may result from misregulation of the APC/C destruction pathway. This profile of misregulated mitotic APC/C substrates and regulators in malignant tumors suggests that analysis of this pathway may be diagnostically useful and represent a potentially important therapeutic target.

Abstract

Vertebrate eggs arrest at metaphase of meiosis II due to an activity known as cytostatic factor (CSF). CSF antagonizes the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C), preventing cyclin B destruction and meiotic exit until fertilization occurs. A puzzling feature of CSF arrest is that APC/C inhibition is leaky. Ongoing cyclin B synthesis is counterbalanced by a limited amount of APC/C-mediated cyclin B destruction; thus, cyclin B/Cdc2 activity remains at steady state. How the APC/C can be slightly active toward cyclin B, and yet restrained from ubiquitinating cyclin B altogether, is unknown. Emi2/XErp1 is the critical CSF component directly responsible for APC/C inhibition during CSF arrest. Fertilization triggers the Ca2+-dependent destruction of Emi2, releasing the APC/C to ubiquitinate the full pool of cyclin B and initiate completion of meiosis. Previously, we showed that a phosphatase maintains Emi2's APC/C-inhibitory activity in CSF-arrested Xenopus egg extracts. Here, we demonstrate that phosphatase inhibition permits Emi2 phosphorylation at thr-545 and -551, which inactivates Emi2. Furthermore, we provide evidence that adding excess cyclin B to CSF extracts stimulates Cdc2 phosphorylation of these same residues, antagonizing Emi2-APC/C association. Our findings suggest a model wherein the pool of Emi2 acts analogously to a rheostat by integrating Cdc2 and phosphatase activities to prevent cyclin B overaccumulation and Cdc2 hyperactivity during the indefinite period of time between arrival at metaphase II and eventual fertilization. Finally, we propose that inactivation of Emi2 by Cdc2 permits mitotic progression during early embryonic cleavage cycles.

Abstract

Cytostatic factor (CSF) arrests unfertilized vertebrate eggs in metaphase of meiosis II by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from mediating cyclin destruction. The APC/C inhibitor Emi2/XErp1 satisfies a number of historical criteria for the molecular identification of CSF, but the mechanism by which CSF is activated selectively in meiosis II is the remaining unexplained criterion. Here we provide an explanation by showing that Emi2 is expressed specifically in meiosis II through translational de-repression or "unmasking" of its mRNA. We find that Emi2 protein is undetectable in immature, G2/prophase-arrested Xenopus oocytes and accumulates approximately 90 minutes after germinal vesicle breakdown. The 3' untranslated region of Emi2 mRNA contains cytoplasmic polyadenylation elements that directly bind the CPEB protein and confer temporal regulation of Emi2 polyadenylation and translation. Our results demonstrate that cytoplasmic polyadenylation and translational unmasking of Emi2 directs meiosis II-specific CSF arrest.

Abstract

Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood.We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions.Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.

Abstract

The tumor suppressor function of the retinoblastoma protein (pRb) is historically attributed to inhibition of E2F gene transcription. In a recent issue of Nature Cell Biology, Binné and colleagues show that pRB is physically linked to the active anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, suggesting that pRB-mediated tumor suppression may also function by directing the local degradation of E2F targets, including Skp2 (Binné et al., 2006).

Identification of Rab11 as a small GTPase binding protein for the Evi5 oncogenePROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAWestlake, C. J., Junutula, J. R., Simon, G. C., Pilli, M., Prekeris, R., Scheller, R. H., Jackson, P. K., Eldridge, A. G.2007; 104 (4): 1236-1241

Abstract

The Evi5 oncogene has recently been shown to regulate the stability and accumulation of critical G(1) cell cycle factors including Emi1, an inhibitor of the anaphase-promoting complex/cyclosome, and cyclin A. Sequence analysis of the amino terminus of Evi5 reveals a Tre-2, Bub2, Cdc16 domain, which has been shown to be a binding partner and GTPase-activating protein domain for the Rab family of small Ras-like GTPases. Here we describe the identification of Evi5 as a candidate binding protein for Rab11, a GTPase that regulates intracellular transport and has specific roles in endosome recycling and cytokinesis. By yeast two-hybrid analysis, immunoprecipitation, and Biacore analysis, we demonstrate that Evi5 binds Rab11a and Rab11b in a GTP-dependent manner. However, Evi5 displays no activation of Rab11 GTPase activity in vitro. Evi5 colocalizes with Rab11 in vivo, and overexpression of Rab11 perturbs the localization of Evi5, redistributing it into Rab11-positive recycling endosomes. Interestingly, in vitro binding studies show that Rab11 effector proteins including FIP3 compete with Evi5 for binding to Rab11, suggesting a partitioning between Rab11-Evi5 and Rab11 effector complexes. Indeed, ablation of Evi5 by RNA interference causes a mislocalization of FIP3 at the abscission site during cytokinesis. These data demonstrate that Evi5 is a Rab11 binding protein and that Evi5 may cooperate with Rab11 to coordinate vesicular trafficking, cytokinesis, and cell cycle control independent of GTPase-activating protein function.

Abstract

Mammalian oocytes are arrested in prophase of the first meiotic division. Progression into the first meiotic division is driven by an increase in the activity of maturation-promoting factor (MPF). In mouse oocytes, we find that early mitotic inhibitor 1 (Emi1), an inhibitor of the anaphase-promoting complex (APC) that is responsible for cyclin B destruction and inactivation of MPF, is present at prophase I and undergoes Skp1-Cul1-F-box/betaTrCP-mediated destruction immediately after germinal vesicle breakdown (GVBD). Exogenous Emi1 or the inhibition of Emi1 destruction in prophase-arrested oocytes leads to a stabilization of cyclin B1-GFP that is sufficient to trigger GVBD. In contrast, the depletion of Emi1 using morpholino oligonucleotides increases cyclin B1-GFP destruction, resulting in an attenuation of MPF activation and a delay of entry into the first meiotic division. Finally, we show that Emi1-dependent effects on meiosis I require the presence of Cdh1. These observations reveal a novel mechanism for the control of entry into the first meiotic division: an Emi1-dependent inhibition of APC(Cdh1).

Abstract

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1. Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.

Abstract

The periodic destruction of mitotic cyclins is triggered by the activation of the anaphase-promoting complex/cyclosome (APC/C) in mitosis. Although the ability of the APC/C to recognize destruction box (D-box) substrates oscillates throughout the cell cycle, the mechanism regulating APC/C binding to D-box substrates remains unclear. Here, we show that the APC/C inhibitor Emi1 tightly binds both the APC/C and its Cdh1 activator, binds to the D-box receptor site on the APC/C(Cdh1), and competes with APC/C substrates for D-box binding. Emi1 itself contains a conserved C-terminal D-box, which provides APC/C-binding affinity, and a conserved zinc-binding region (ZBR), which antagonizes APC/C E3 ligase activity independent of tight APC binding. Mutation of the ZBR converts Emi1 into a D-box-dependent APC/C substrate. The identification of a direct Emi1-APC/C complex further explains how Emi1 functions as a stabilizing factor for cyclin accumulation and the need to destroy Emi1 for APC/C activation in mitosis. The combination of a degron/E3 recognition site and an anti-ligase function in Emi1 suggests a general model for how E3 substrates evolve to become pseudosubstrate inhibitors.

Abstract

The anaphase promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that controls the cell cycle by directing the ubiquitin-dependent proteolysis of S-phase and mitosis promoting factors. Emi1 is an E2F transcriptional target that drives cell cycle progression from G1/S through early mitosis by inhibiting the APC/C's ubiquitin ligase activity, and thus facilitates accumulation of APC/C substrates. Using cell culture model systems, we found that Emi1 overexpression leads to proliferation, tetraploidy and genome instability of cells deficient for p53. We propose that loss of pRb repression of E2F-mediated transcription causing misregulation of Emi1 and APC/C substrates results in the generation of tetraploidy and proliferation of genomically unstable cells in the absence of normal p53 function. This represents a potentially important mechanism by which pRb and p53 dysfunction may contribute to tumorigenesis through the generation of genomic instability.

Abstract

Greatwall kinase is a conserved regulator of mitotic entry, and new work in Xenopus egg extracts (Yu et al., 2006) shows that Greatwall is required for the positive feedback loop that removes inhibitory tyrosine phosphate from the central mitotic regulatory kinase Cdc2.

Abstract

Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, D(N) and D(C), and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy.

CaMKII and Polo-like kinase 1 sequentially phosphorylate the cytostatic factor Emi2/XErp1 to trigger its destruction and meiotic exitPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAHansen, D. V., Tung, J. J., Jackson, P. K.2006; 103 (3): 608-613

Abstract

In vertebrate meiosis, unfertilized eggs are arrested in metaphase II by cytostatic factor (CSF), which is required to maintain mitotic cyclin-dependent kinase activity. Fertilization triggers a transient increase in cytosolic free Ca(2+), which leads to CSF inactivation and ubiquitin-dependent cyclin destruction through the anaphase promoting complex or cyclosome (APC/C). The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the Polo-like kinase Plx1 are essential factors for Ca(2+)-induced meiotic exit, but the critical targets of these kinases were unknown. The APC/C inhibitor Emi2 or XErp1 has recently been characterized as a pivotal CSF component, required to maintain metaphase II arrest and rapidly destroyed in response to Ca(2+) signaling through phosphorylation by Plx1 and ubiquitination by the SCF(betaTrCP) complex. An important question is how the increase in free Ca(2+) targets Plx1 activity toward Emi2. Here, we demonstrate that CaMKII is required for Ca(2+)-induced Emi2 destruction, and that CaMKII functions as a "priming kinase," directly phosphorylating Emi2 at a specific motif to induce a strong interaction with the Polo Box domain of Plx1. We show that the strict requirement for CaMKII to phosphorylate Emi2 is a specific feature of CSF arrest, and we also use phosphatase inhibitors to demonstrate an additional mode of Emi2 inactivation independent of its destruction. We firmly establish the CSF component Emi2 as the first-known critical and direct target of CaMKII in CSF release, providing a detailed molecular mechanism explaining how CaMKII and Plx1 coordinately direct APC/C activation and meiotic exit upon fertilization.

Abstract

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

A role for the anaphase-promoting complex inhibitor Emi2/XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of Xenopus eggsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICATung, J. J., Hansen, D. V., Ban, K. H., Loktev, A. V., Summers, M. K., Adler, J. R., Jackson, P. K.2005; 102 (12): 4318-4323

Abstract

Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCF(betaTrCP) ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.

Abstract

Expression of the agouti signaling protein (ASIP) during hair growth produces the red/yellow pigment pheomelanin. ASIP, and its neuropeptide homolog the agouti-related protein (AgRP) involved in energy balance, are novel, paracrine signaling molecules that act as inverse agonists at distinct subsets of melanocortin receptors. Ubiquitous ASIP expression in mice gives rise to a pleiotropic phenotype characterized by a uniform yellow coat color, obesity, overgrowth, and metabolic derangements similar to type II diabetes in humans. Here we report the synthesis and NMR structure of ASIP's active, cysteine-rich, C-terminal domain. ASIP adopts the inhibitor cystine knot fold and, along with AgRP, are the only known mammalian proteins in this structure class. Moreover, ASIP populates two distinct conformers resulting from a cis peptide bond at Pro102-Pro103 and a coexistence of cis/trans isomers of Ala104-Pro105. Pharmacologic studies of Pro-->Ala mutants demonstrate that the minor conformation with two cis peptide bonds is responsible for activity at all MCRs. The loop containing the heterogeneous Ala-Pro peptide bond is conserved in mammals, and suggests that ASIP is either trapped by evolution in this unusual configuration or possesses function outside of strict MCR antagonism.

Abstract

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified Delta90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of nondestructible Delta90 cyclin B rescues the MI-MII transition in Emi1-inhibited oocytes.

Abstract

The turnover of key proteins that mediate development, cellular proliferation, and a host of essential biological processes is controlled by the ubiquitin proteasome system (UPS). In several well-studied examples, notably in the cell cycle, regulatory proteins that control ubiquitin-dependent destruction are themselves substrates of the UPS, creating a multilayered system to ensure precise and dynamic control of protein stability. UPS regulators controlled at the level of protein stability--including the F-box protein Skp2 and the VHL protein (substrate adapter proteins for multicomponent E3 ubiquitin ligases)-- seem to be misregulated in tumors. In these cases, especially, measuring levels of critical regulatory and target proteins will often present a more biologically meaningful picture than examining relative mRNA levels, which do not always reflect corresponding protein levels. Tissue microarrays (TMAs) allow simultaneous screening of large numbers of tumors for expression of specific proteins by immunohistochemical staining of a single microscope slide prepared from a TMA paraffin block. Replicate slides prepared from the same block can be immunostained for multiple proteins functioning in a related pathway, and a semiquantitative protein expression profile for a given subset of UPS pathway components, or other subsets of proteins of interest, can be assembled. Protein expression profiles of individual tumors or tissue types can be compared and visualized by hierarchical clustering methods. These expression profiles may be used as screening tools to investigate the relative abundance of components of a biochemical pathway in tumors or other tissues. TMAs have an exciting future as tools for basic research, diagnostic pathology, and drug targeting. In this article, we provide an introduction to the use of TMAs to study the expression of UPS component proteins and substrates in tumors by immunohistochemistry.

Abstract

Progression through mitosis requires activation of cyclin B/Cdk1 and its downstream targets, including Polo-like kinase and the anaphase-promoting complex (APC), the ubiquitin ligase directing degradation of cyclins A and B. Recent evidence shows that APC activation requires destruction of the APC inhibitor Emi1. In prophase, phosphorylation of Emi1 generates a D-pS-G-X-X-pS degron to recruit the SCF(betaTrCP) ubiquitin ligase, causing Emi1 destruction and allowing progression beyond prometaphase, but the kinases directing this phosphorylation remain undefined. We show here that the polo-like kinase Plk1 is strictly required for Emi1 destruction and that overexpression of Plk1 is sufficient to trigger Emi1 destruction. Plk1 stimulates Emi1 phosphorylation, betaTrCP binding, and ubiquitination in vitro and cyclin B/Cdk1 enhances these effects. Plk1 binds to Emi1 in mitosis and the two proteins colocalize on the mitotic spindle poles, suggesting that Plk1 may spatially control Emi1 destruction. These data support the hypothesis that Plk1 activates the APC by directing the SCF-dependent destruction of Emi1 in prophase.

Abstract

The family of cyclin-dependent kinases (Cdks) lies at the core of the machinery that drives the cell division cycle. Studies in cultured mammalian cells have provided insight into the cellular functions of many Cdks. Recent Cdk and cyclin knockouts in the mouse show that the functions of G1 cell cycle regulatory genes are often essential only in specific cell types, pointing to our limited understanding of tissue-specific expression, redundancy, and compensating mechanisms in the Cdk network.

Abstract

The dual specificity phosphatase Cdc14 has been shown to be a critical regulator of late mitotic events in several eukaryotes, including S. cerevisiae, S. pombe. C. elegans and H. sapiens. However, Cdc14 homologs have clearly evolved to regulate distinct cellular processes and to respond to regulatory signals important for these processes. The human paralogs hCdc14A and B are the only vertebrate Cdc14 homologues studied to date, but their functions are not well understood. Therefore, it is of great interest to examine the function Cdc14 homologs in other vertebrate species.We identified two open reading frames from Xenopus laevis closely related to human Cdc14A, called XCdc14alpha and XCdc14beta, although no obvious paralog of the hCdc14B was found. To begin a functional characterization of Xcdc14alpha and XCdc14beta, we raised polyclonal antibodies against a conserved region. These antibodies stained both the nucleolus and centrosome in interphase Xenopus tissue culture cells, and the mitotic centrosomes. GFP-tagged version of XCdc14alpha localized to the nucleulus and GFP-XCdc14beta localized to the centrosome, although not exclusively. XCdc14alpha was also both meiotically and mitotically phosphorylated. Injection of antibodies raised against a conserved region of XCdc14/beta into Xenopus embryos at the two-cell stage blocked division of the injected blastomeres, suggesting that activities of XCdc14alpha/beta are required for normal cell division.These results provide evidence that XCdc14alpha/beta are required for normal cellular division and are regulated by at least two mechanisms, subcellular localization and possibly phosphorylation. Due to the high sequence conservation between Xcdc14alpha and hCdc14A, it seems likely that both mechanisms will contribute to regulation of Cdc14 homologs in vertebrates.

Abstract

A recent study shows that the RASSF1A tumor suppressor functions as a regulator of the ordered proteolytic steps that organize mitosis. By controlling the stability of microtubules and the activity of the anaphase-promoting complex (APC), RASSF1A might provide a crucial link between mechanisms of tumor suppression and mitotic cell division. Furthermore, another recent study shows that protein kinase A, which is a key growth regulator, inhibits the APC during mitosis in yeast.

Abstract

Progression through mitosis occurs because cyclin B/Cdc2 activation induces the anaphase promoting complex (APC) to cause cyclin B destruction and mitotic exit. To ensure that cyclin B/Cdc2 does not prematurely activate the APC in early mitosis, there must be a mechanism delaying APC activation. Emi1 is a protein capable of inhibiting the APC in S and G2. We show here that Emi1 is phosphorylated by Cdc2, and on a DSGxxS consensus site, is subsequently recognized by the SCF(betaTrCP/Slimb) ubiquitin ligase and destroyed, thus providing a delay for APC activation. Failure of betaTrCP-dependent Emi1 destruction stabilizes APC substrates and results in mitotic catastrophe including centrosome overduplication, potentially explaining mitotic deficiencies in Drosophila Slimb/betaTrCP mutants. We hypothesize that Emi1 destruction relieves a late prophase checkpoint for APC activation.

Abstract

SCF ubiquitin ligases, composed of three major subunits, Skp1, Cul1, and one of many F box proteins (Fbps), control the proteolysis of important cellular regulators. We have inactivated the gene encoding the Fbp beta-Trcp1 in mice. beta-Trcp1(-/-) males show reduced fertility correlating with an accumulation of methaphase I spermatocytes. beta-Trcp1(-/-) MEFs display a lengthened mitosis, centrosome overduplication, multipolar metaphase spindles, and misaligned chromosomes. Furthermore, cyclin A, cyclin B, and Emi1, an inhibitor of the anaphase promoting complex, are stabilized in mitotic beta-Trcp1(-/-) MEFs. Indeed, we demonstrate that Emi1 is a bona fide substrate of beta-Trcp1. In contrast, stabilization of beta-catenin and IkappaBalpha, two previously reported beta-Trcp1 substrates, does not occur in the absence of beta-Trcp1 and instead requires the additional silencing of beta-Trcp2 by siRNA. Thus, beta-Trcp1 regulates the timely order of meiotic and mitotic events.

Abstract

mahoganoid is a mouse coat-color mutation whose pigmentary phenotype and genetic interactions resemble those of Attractin (Atrn). Atrn mutations also cause spongiform neurodegeneration. Here, we show that a null mutation for mahoganoid causes a similar age-dependent neuropathology that includes many features of prion diseases but without accumulation of protease-resistant prion protein. The gene mutated in mahoganoid encodes a RING-containing protein with E3 ubiquitin ligase activity in vitro. Similarities in phenotype, expression, and genetic interactions suggest that mahoganoid and Atrn genes are part of a conserved pathway for regulated protein turnover whose function is essential for neuronal viability.

Abstract

Substrate binding by the SCFCdc4 ubiquitin ligase is regulated by phosphorylation. In this issue of Cell, Orlicky et al. describe the crystal structure of the Cdc4 subunit bound to a high-affinity substrate phosphopeptide. This structure provides insights into the binding interaction and how a precise mechanism involving multiple regulatory phosphorylations may be mediated by a single binding site.

Abstract

Switching from eumelanin to pheomelanin synthesis during hair growth is accomplished by transient synthesis of Agouti protein, an inverse agonist for the melanocortin-1 receptor (Mc1r). The coat color mutations mahogany and mahoganoid prevent hair follicle melanocytes from responding to Agouti protein. The gene mutated in mahogany, which is also known as Attractin (Atrn), encodes a type I transmembrane protein that functions as an accessory receptor for Agouti protein. We have recently determined that the gene mutated in mahoganoid, which is also known as Mahogunin (Mgrn1), encodes an E3 ubiquitin ligase. Like Attractin, Mahogunin is conserved in invertebrate genomes, and its absence causes a pleiotropic phenotype that includes spongiform neurodegeneration.

Abstract

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. We recently determined the NMR structure of AGRP(87-132) and demonstrated that a portion of the domain adopts the inhibitor cystine-knot fold. Remarkably, this is the first identification of a mammalian protein with this specific architecture. Further analysis of the structure suggests that melanocortin receptor contacts are made primarily by two loops presented within the cystine knot. (10) To test this hypothesis we designed a 34-residue AGRP analogue corresponding to only the cystine knot. We found that this designed miniprotein folds to a homogeneous product, retains the desired cystine-knot architecture, functions as a potent antagonist, and maintains the melanocortin receptor pharmacological profile of AGRP(87-132). (26) The AGRP-like activity of this molecule supports the hypothesis that indeed the cystine-knot region possesses the melanocortin receptor contacts. Based on these design and structure studies, we propose that the N-terminal loop of AGRP(87-132) makes contact with a receptor exoloop and helps confer AGRP's selectivity for the central MCRs.

Abstract

We have identified a family of RING finger proteins that are orthologous to Drosophila Goliath (G1, Gol). One of the members, GREUL1 (Goliath Related E3 Ubiquitin Ligase 1), can convert Xenopus ectoderm into XAG-1- and Otx2-expressing cells in the absence of both neural tissue and muscle. This activity, combined with the finding that XGREUL1 is expressed within the cement gland, suggests a role for GREUL1 in the generation of anterior ectoderm. Although GREUL1 is not a direct inducer of neural tissue, it can activate the formation of ectopic neural cells within the epidermis of intact embryos. This suggests that GREUL1 can sensitize ectoderm to neuralizing signals. In this paper, we provide evidence that GREUL1 is an E3 ubiquitin ligase. Using a biochemical assay, we show that GREUL1 catalyzes the addition of polyubiquitin chains. These events are mediated by the RING domain since a mutation in two of the cysteines abolishes ligase activity. Mutation of these cysteines also compromises GREUL1's ability to induce cement gland. Thus, GREUL1's RING domain is necessary for both the ubiquitination of substrates and for the conversion of ectoderm to an anterior fate.

Abstract

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and gamma-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

Abstract

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCF(Skp2) ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished.

Abstract

Emi1 promotes mitotic entry in Xenopus laevis embryos by inhibiting the APC(Cdc20) ubiquitination complex to allow accumulation of cyclin B. We show here that human Emi1 (hEmi1) functions to promote cyclin A accumulation and S phase entry in somatic cells by inhibiting the APC(Cdh1) complex. At the G1-S transition, hEmi1 is transcriptionally induced by the E2F transcription factor, much like cyclin A. hEmi1 overexpression accelerates S phase entry and can override a G1 block caused by overexpression of Cdh1 or the E2F-inhibitor p105 retinoblastoma protein (pRb). Depleting cells of hEmi1 through RNA interference prevents accumulation of cyclin A and inhibits S phase entry. These data suggest that E2F can activate both transcription of cyclin A and the hEmi1-dependent stabilization of APC(Cdh1) targets, such as cyclin A, to promote S phase entry.

Abstract

Vertebrate eggs are arrested at metaphase of meiosis II with stable cyclin B and high cyclin B/Cdc2 kinase activity. The ability of the anaphase-promoting complex/cyclosome (APC), an E3 ubiquitin ligase, to trigger cyclin B destruction and metaphase exit is blocked in eggs by the activity of cytostatic factor (CSF) (reviewed in ref. 1). CSF was defined as an activity in mature oocytes that caused mitotic arrest when injected into dividing embryos. Fertilization causes a transient increase in cytoplasmic calcium concentration leading to CSF inactivation, APC activation, cyclin B destruction and mitotic exit. The APC activator Cdc20 is required for APC activation after fertilization. We show here that the APC(cdc20) inhibitor Emi1 (ref. 6) is necessary and sufficient to inhibit the APC and to prevent mitotic exit in CSF-arrested eggs. CSF extracts immunodepleted of Emi1 degrade cyclin B, and exit from mitosis prematurely in the absence of calcium. Addition of Emi1 to these Emi1-depleted extracts blocks premature inactivation of the CSF-arrested state. Emi1 is required to arrest unfertilized eggs at metaphase of meiosis II and seems to be the long-sought mediator of CSF activity.

Abstract

We show that human Cdc14A phosphatase interacts with interphase centrosomes, and that this interaction is independent of microtubules and Cdc14A phosphatase activity, but requires active nuclear export. Disrupting the nuclear export signal (NES) led to Cdc14A being localized in nucleoli, which in unperturbed cells selectively contain Cdc14B (ref. 1). Conditional overproduction of Cdc14A, but not its phosphatase-dead or NES-deficient mutants, or Cdc14B, resulted in premature centrosome splitting and formation of supernumerary mitotic spindles. In contrast, downregulation of endogenous Cdc14A by short inhibitory RNA duplexes (siRNA) induced mitotic defects including impaired centrosome separation and failure to undergo productive cytokinesis. Consequently, both overexpression and downregulation of Cdc14A caused aberrant chromosome partitioning into daughter cells. These results indicate that Cdc14A is a physiological regulator of the centrosome duplication cycle, which, when disrupted, can lead to genomic instability in mammalian cells.

Abstract

The anaphase-promoting complex/cyclosome (APC) ubiquitin ligase is activated by Cdc20 and Cdh1 and inhibited by Mad2 and the spindle assembly checkpoint complex, Mad2B, and the early mitotic inhibitor Emi1. Mad2 inhibits APC(Cdc20), whereas Mad2B preferentially inhibits APC(Cdh1). We have examined the mechanism of APC inhibition by Emi1 and find that unlike Mad2 proteins, Emi1 binds and inhibits both APC(Cdh1) and APC(Cdc20). Also unlike Mad2, Emi1 stabilizes cyclin A in the embryo and requires zinc for its APC inhibitory activity. We find that Emi1 binds the substrate-binding region of Cdc20 and prevents substrate binding to the APC, illustrating a novel mechanism of APC inhibition.

Abstract

To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.

Abstract

Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.

Abstract

Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.

Nuclear accumulation of cyclin E/Cdk2 triggers a concentration-dependent switch for the destruction of p27 (Xic1)PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICASwanson, C., Ross, J., Jackson, P. K.2000; 97 (14): 7796-7801

Abstract

The action of cyclin-dependent kinases (CDKs) is regulated by phosphorylation, cyclin levels, the abundance of CDK inhibitors, and, as recently has been shown for cyclin B/cdc2, their localization. It is unclear how localization regulates the action of cyclin E/Cdk2 and its inhibitors. Here, we show that the closest known Xenopus laevis homolog of mammalian Cdk2 inhibitors p27(Kip1) and p21(CIP1), Xic1, is concentrated, ubiquitinated, and destroyed in the nucleus. Furthermore, Xic1 destruction requires nuclear import, but not nuclear export, and requires the formation of a transport-competent nuclear envelope, but not interactions between the lamina and chromatin. We show that (i) cyclin E/Cdk2 and Xic1 are transported into the nucleus as a complex and that Xic1 destruction requires the activity of cyclin E, (ii) that phosphorylation of Xic1 by cyclin E/Cdk2 bypasses the requirement for nuclear formation, and (iii) that the phosphorylation of Xic1 by cyclin E/Cdk2 is concentration dependent and likely realized through second-order interactions between stable cyclin E/Cdk2/Xic1 ternary complexes. Based on these results we propose a model wherein nuclear accumulation of the cyclin E/Cdk2/Xic1 complex triggers a concentration-dependent switch that promotes the phosphorylation of Xic1 and, consequently, its ubiquitination and destruction, thus allowing subsequent activation of cyclin E/Cdk2.

Abstract

In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce rereplication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF(Pop) ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins.

Abstract

Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.

Abstract

Ubiquitin-dependent proteolysis plays an important role in cell-cycle control [1] [2]. In budding yeast, the protein Skp1p, the cullin-family member Cdc53p, and the F-box/WD-repeat protein Cdc4p form the SCFCdc4p ubiquitin ligase complex, which targets the cyclin-dependent kinase (Cdk) inhibitor Sic1p for proteolysis [3] [4] [5] [6] [7] [8]. Sic1p is recruited to the SCFCdc4p complex by binding to the WD-repeat region of Cdc4p [5] [6], while Skp1p binds to the F-box of Cdc4p [9]. In fission yeast, two distinct Cdc4p-related proteins, Pop1p/Ste16p [10] [11] and the recently identified Sud1p/Pop2p [12], regulate the stability of the replication initiator Cdc18p and the Cdk inhibitor Rum1p. We show here that, despite their structural and functional similarities, the pop1 and pop2 genes fail to complement each other's deletion phenotypes, indicating that they perform non-redundant, but potentially interdependent, functions in proteolysis. Consistent with this hypothesis, Pop1p and Pop2p formed heterooligomeric complexes when overexpressed, and binding of Cdc18p to Pop2p was dependent on Pop1p. The Pop1p-Pop2p interaction was mediated by the amino-terminal domain of Pop2p which, when fused to full-length Pop1p, rescued the phenotype of a Deltapop1Deltapop2 double mutant. Thus, close physical proximity of two distinct F-box/WD-repeat proteins directs proteolysis mediated by the SCFPop ubiquitin ligase complex.

Cyclin-dependent kinase control of centrosome duplicationPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALacey, K. R., Jackson, P. K., Stearns, T.1999; 96 (6): 2817-2822

Abstract

Centrosomes nucleate microtubules and duplicate once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome/cell ratio. Centrosome duplication occurs during the G1/S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitoses. Using both in vivo and in vitro assays, we show that centrosome duplication in Xenopus laevis embryos requires cyclin/cdk2 kinase activity. Injection of the cdk (cyclin-dependent kinase) inhibitor p21 into one blastomere of a dividing embryo blocks centrosome duplication in that blastomere; the related cdk inhibitor p27 has a similar effect. An in vitro system using Xenopus extracts carries out separation of the paired centrioles within the centrosome. This centriole separation activity is dependent on cyclin/cdk2 activity; depletion of either cdk2 or of the two activating cyclins, cyclin A and cyclin E, eliminates centriole separation activity. In addition, centriole separation is inhibited by the mitotic state, suggesting a mechanism of linking the cell cycle to periodic duplication of the centrosome.

Abstract

The anaphase-promoting complex (APC) or cyclosome directs the ubiquitination and destruction of proteins that control specific steps in mitosis. Recent studies show that APC activity requires WD40 domain proteins, and that one of these proteins is part of the checkpoint control that ensures accurate chromosome segregation.

Abstract

A newly discovered family of proteins homologous to yeast Cdc53, called cullins, may play a key role in the targeting of cell-cycle regulators, such as cyclins, for destruction by ubiquitin-dependent proteolysis.

Abstract

c-Abl is a non-receptor protein-tyrosine kinase lacking a clear physiological role. A clue to its normal function is suggested by overexpression of Abl in fibroblasts, which leads to inhibition of cell growth. This effect requires tyrosine kinase activity and the Abl C-terminus. c-Abl is localized to the cell nucleus, where it can bind DNA, and interacts with the retinoblastoma protein, a potential mediator of the growth-inhibitory effect. Nuclear localization of Abl can be directed by a pentalysine nuclear localization signal in the Abl C-terminus. Here, we have identified two additional basic motifs in the Abl C-terminus, either of which can function independently of the pentalysine signal to localize Abl to the nucleus. Using a quantitative transfection assay, we show that both c-Abl and transforming Abl proteins inhibit entry into S phase and this effect is absolutely dependent on nuclear localization. Further, we demonstrate that the Abl cytostatic effect requires both the Rb and p53 tumor suppressor gene products. These results indicate that Abl inhibits cell proliferation by interacting with central elements of the cell cycle control apparatus in the nucleus, and suggest a direct connection between p53 and Rb in this growth-inhibitory pathway.

Abstract

Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle.

Abstract

Activated forms of the nuclear and cytoplasmic tyrosine kinase c-Abl are completely cytoplasmic and oncogenic. The overexpression of c-Abl, and in certain fibroblast cell lines even of v-Abl, leads to a cell cycle arrest revealing an alternative Abl function. To facilitate the analysis of this growth inhibitory function we have taken advantage of regulable Abl-estrogen receptor (ABL:ER) fusion proteins. Oncogenic in the presence of estrogen, they are reversibly switched to inhibit cell proliferation upon removal of hormone. Using this system, we demonstrate that inhibition is effected by Abl derivatives which we have previously shown to be hypo-phosphorylated and to have low kinase activity. Since an almost exclusively cytoplasmic ABL:ER protein is fully growth inhibitory, relevant interactions may occur in the cytoplasm. We identify the cell cycle arrest as an early G1 or G0-like block. Interestingly, growth inhibition correlates with an altered expression pattern of early serum response genes; c-Jun mRNA and c-Fos protein levels are elevated in Abl-blocked cells. In view of the two functional modes of overexpressed Abl proteins, one can speculate that normal c-Abl may be involved in relaying growth regulatory signals from the membrane to the nucleus.

Abstract

The protein p21 (WAF1, CIP1 or sdi1), induced by the tumour-suppressor protein p53, interacts with and inhibits two different targets essential for cell-cycle progression. One of these is the cyclin-Cdk family of kinases and the other is the essential DNA replication factor, proliferating-cell nuclear antigen (PCNA). We report here that separate domains of p21 are responsible for interacting with and inhibiting the two targets. An amino-terminal domain inhibits cyclin-Cdk kinases and a carboxy-terminal domain inhibits PCNA. Using these separated domains, we have determined that p21 inhibits different biological systems through different targets. The PCNA-binding domain is sufficient for inhibition of DNA replication based on simian virus 40, whereas the Cdk2-binding domain is sufficient for inhibition of DNA replication based on Xenopus egg extract and for growth suppression in transformed human cells.

Abstract

The myristoylated form of c-Abl protein, as well as the P210bcr/abl protein, have been shown by indirect immunofluorescence to associate with F-actin stress fibers in fibroblasts. Analysis of deletion mutants of c-Abl stably expressed in fibroblasts maps the domain responsible for this interaction to the extreme COOH-terminus of Abl. This domain mediates the association of a heterologous protein with F-actin filaments after microinjection into NIH 3T3 cells, and directly binds to F-actin in a cosedimentation assay. Microinjection and cosedimentation assays localize the actin-binding domain to a 58 amino acid region, including a charged motif at the extreme COOH-terminus that is important for efficient binding. F-actin binding by Abl is calcium independent, and Abl competes with gelsolin for binding to F-actin. In addition to the F-actin binding domain, the COOH-terminus of Abl contains a proline-rich region that mediates binding and sequestration of G-actin, and the Abl F- and G-actin binding domains cooperate to bundle F-actin filaments in vitro. The COOH terminus of Abl thus confers several novel localizing functions upon the protein, including actin binding, nuclear localization, and DNA binding. Abl may modify and receive signals from the F-actin cytoskeleton in vivo, and is an ideal candidate to mediate signal transduction from the cell surface and cytoskeleton to the nucleus.

Abstract

Fusion of the hormone binding domain (HBD) of steroid receptors to transcription factors renders them hormone-dependent. We show here that an SH3-deleted, oncogenic variant of the Abl tyrosine kinase becomes hormone-dependent for transformation by fusion to the estrogen receptor (ER) HBD, extending the phenomenon to tyrosine kinases. Surprisingly, fusion of the HBD to the normal, non-transforming c-Abl (IV) protein activated transforming activity in a hormone-dependent fashion. In the presence of hormone, the c-Abl:ER fusion protein was transforming, cytoplasmic and tyrosine phosphorylated, whereas it was non-transforming, nuclear and hypophosphorylated without hormone. We have examined the kinetics of activation of the c-Abl:ER protein and found that protein synthesis is required both for kinase activation and for redistribution of the c-Abl:ER protein from the nucleus to the cytoplasm. We suggest that the activation of c-Abl could be due to HBD-mediated dimerization and/or to the ability to overexpress conditionally the normally toxic c-Abl protein. This novel approach may be applicable to a wide variety of proteins, particularly when activating mutations or physiological inducers are unknown or when the protein is toxic to cells.

Abstract

c-abl is the normal cellular homolog of the v-abl transforming gene of Abelson murine leukemia virus. By constructing recombinants between c- and v-abl retroviruses, we show that a point mutation in c-Abl is sufficient to change the myristoylated form of c-Abl into a protein able to transform fibroblasts, but not capable of transforming bone marrow or inducing Abelson disease. This activating mutation, which changes the phenylalanine at amino acid 420 to valine (F420V) found in the homologous position of v-Abl, is positioned outside of the SH3 domain, a region typically modified in transforming alleles of abl. Phenylalanine 420 is perfectly conserved among tyrosine kinases with N-terminal SH3 domains (the Src and Abl families). The equivalent position in other protein tyrosine kinases is a conserved hydrophobic residue that predicts the specific family to which that kinase belongs. Mutation of phenylalanine 420 to other hydrophobic residues activates c-Abl. Unlike other transforming variants of Abl, the F420V mutant protein is not highly phosphorylated on tyrosine. Mutation of the nearby proposed autophosphorylation site, tyrosine 412, shows that this tyrosine is not strictly required for fibroblast transformation in either F420V or SH3-deleted variants of c-Abl (IV).

Abstract

N-terminal myristoylation can promote the association of proteins with the plasma membrane, a property that is required for oncogenic variants of Src and Abl to transform fibroblastic cell types. The P210bcr/abl protein of chronic myelogenous leukemia cells is not myristoylated and does not stably transform NIH 3T3 fibroblasts; however, it will transform lymphoid and myeloid cell types in vitro and in vivo, suggesting that myristoylation is not required for Abl variants to transform hematopoietic cells. To test this hypothesis, we introduced point mutations that disrupt myristoylation into two activated Abl proteins, v-Abl and a deletion mutant of c-Abl (delta XB), and examined their ability to transform an interleukin-3-dependent lymphoblastoid cell line, Ba/F3. Neither of the nonmyristoylated Abl proteins transformed NIH 3T3 fibroblasts, but like P210bcr/abl, both were capable of transforming the Ba/F3 cells to factor independence and tumorigenicity. Nonmyristoylated Abl variants did not associate with the plasma membrane in the transformed Ba/F3 cells. These results demonstrate that Abl proteins can transform hematopoietic cells in the absence of membrane association and suggest that distinct functions of Abl are required for transformation of fibroblast and hematopoietic cell types.

Abstract

We have constructed a series of point mutations in the highly conserved FLVRES motif of the src homology 2 (SH2) domain of the abl tyrosine kinase. Mutant SH2 domains were expressed in bacteria, and their ability to bind to tyrosine-phosphorylated proteins was examined in vitro. Three mutants were greatly reduced in their ability to bind both phosphotyrosine itself and tyrosine-phosphorylated cellular proteins. All of the mutants that retained activity bound to the same set of tyrosine-phosphorylated proteins as did the wild type, suggesting that binding specificity was unaffected. These results implicate the FLVRES motif in direct binding to phosphotyrosine. When the mutant SH2 domains were inserted into an activated abl kinase and expressed in murine fibroblasts, decreased in vitro phosphotyrosine binding correlated with decreased transforming ability. This finding implies that SH2-phosphotyrosine interactions are involved in transmission of positive growth signals by the nonreceptor tyrosine kinases, most likely via the assembly of multiprotein complexes with other tyrosine-phosphorylated proteins.

Abstract

The c-abl proto-oncogene, which encodes a cytoplasmic protein-tyrosine kinase, is expressed throughout murine gestation and ubiquitously in adult mouse tissues. However, its levels are highest in thymus, spleen, and testes. To examine the in vivo role of c-abl, the gene was disrupted in embryonic stem cells, and the resulting genetically modified cells were used to establish a mouse strain carrying the mutation. Most mice homozygous for the c-abl mutation became runted and died 1 to 2 weeks after birth. In addition, many showed thymic and splenic atrophy and a T and B cell lymphopenia.

THE NONCATALYTIC SRC HOMOLOGY REGION-2 SEGMENT OF ABL TYROSINE KINASE BINDS TO TYROSINE-PHOSPHORYLATED CELLULAR PROTEINS WITH HIGH-AFFINITYPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMayer, B. J., Jackson, P. K., Baltimore, D.1991; 88 (2): 627-631

Abstract

Several proteins implicated in the regulation of cell proliferation contain a common noncatalytic domain, src homology region 2 (SH2). We have used the bacterially expressed SH2 domain of abl protein-tyrosine kinase to evaluate the ability of this domain to bind to cellular proteins. ablSH2 specifically bound to a number of tyrosine-phosphorylated proteins from cells transformed by tyrosine kinase oncogenes in a filter-binding assay and to a subset of those proteins in solution. The SH2 probe bound almost exclusively to tyrosine-phosphorylated proteins, and binding was eliminated by dephosphorylation of cell proteins. Free phosphotyrosine could partially disrupt SH2 binding, suggesting that phosphotyrosine is directly involved in the binding interaction. These results demonstrate that an SH2 domain is sufficient to confer direct, high-affinity phosphotyrosine-dependent binding to proteins and suggest a general role for SH2 domains in cellular signaling pathways.

Abstract

The subcellular localization of the mouse type IV c-abl protein was determined by indirect immunofluorescence of nontransformed NIH 3T3 fibroblasts that overexpress the protein. Unlike the viral transforming protein p160gag/v-abl, which has cytoplasmic and plasma membrane localization, a large fraction of the c-abl (IV) protein is nuclear, with the remainder in the cytoplasm and plasma membrane. Deletion of a small N-terminal regulatory region of the c-abl (IV) protein, sufficient to activate its transforming potential fully, changes the distribution of the protein from the nucleus to the cytoplasm. Mapping of an amino acid sequence responsible for the nuclear localization of the c-abl (IV) protein reveals a nuclear localization signal similar to that of SV40 large T antigen.

Abstract

The two major forms of the c-abl gene differ from their activated counterpart, the v-abl oncogene of the Abelson murine leukemia virus by the replacement of their N-terminal sequences with viral gag sequences. Overexpression of p150c-abl type IV in a retroviral vector similar to Abelson virus does not transform NIH 3T3 fibroblasts, even though it is expressed and myristoylated at levels comparable to pp160v-abl. Members of a nested set of deletion mutations of the N-terminus of c-abl type IV in this expression system will activate abl to transform murine fibroblasts. The smallest of these deletions, delta XB, efficiently transforms lymphoid cells in vitro and causes leukemia in vivo demonstrating that gag sequences are not necessary for abl-induced leukemogenesis. The delta XB mutation defines an N-terminal regulatory domain, which shares a surprising homology with chicken oncogene v-crk and phospholipase C-II. Although overexpression of the myristoylated form of c-abl does not transform cells, it nonetheless has a profound effect on cell growth.